Estimating the volume of penumbra in rodents using DTI and stack-based ensemble machine learning framework.

BACKGROUND: This study investigates the potential of diffusion tensor imaging (DTI) in identifying penumbral volume (PV) compared to the standard gadolinium-required perfusion-diffusion mismatch (PDM), utilizing a stack-based ensemble machine learning (ML) approach with enhanced explainability. METHODS: Sixteen male rats were subjected to middle cerebral artery occlusion. The penumbra was identified using PDM at 30 and 90 min after occlusion. We used 11 DTI-derived metrics and 14 distance-based features to train five voxel-wise ML models. The model predictions were integrated using stack-based ensemble techniques. ML-estimated and PDM-defined PVs were compared to evaluate model performance through volume similarity assessment, the Pearson correlation analysis, and Bland-Altman analysis. Feature importance was determined for explainability. RESULTS: In the test rats, the ML-estimated median PV was 106.4 mL (interquartile range 44.6-157.3 mL), whereas the PDM-defined median PV was 102.0 mL (52.1-144.9 mL). These PVs had a volume similarity of 0.88 (0.79-0.96), a Pearson correlation coefficient of 0.93 (p < 0.001), and a Bland-Altman bias of 2.5 mL (2.4% of the mean PDM-defined PV), with 95% limits of agreement ranging from -44.9 to 49.9 mL. Among the features used for PV prediction, the mean diffusivity was the most important feature. CONCLUSIONS: Our study confirmed that PV can be estimated using DTI metrics with a stack-based ensemble ML approach, yielding results comparable to the volume defined by the standard PDM. The model explainability enhanced its clinical relevance. Human studies are warranted to validate our findings. RELEVANCE STATEMENT: The proposed DTI-based ML model can estimate PV without the need for contrast agent administration, offering a valuable option for patients with kidney dysfunction. It also can serve as an alternative if perfusion map interpretation fails in the clinical setting. KEY POINTS: • Penumbral volume can be estimated by DTI combined with stack-based ensemble ML. • Mean diffusivity was the most important feature used for predicting penumbral volume. • The proposed approach can be beneficial for patients with kidney dysfunction.

Optogenetic stimulation in the medial prefrontal cortex modulates stimulus valence from rewarding and aversive to neutral states.

Introduction: Understanding the modulations of the medial prefrontal cortex (mPFC) in the valence of the stimulus from rewarding and aversive status to neutral status is crucial for the development of novel treatments for drug addiction. This study addressed this issue and examined whether optogenetic ChR2 photostimulation in the cingulate, prelimbic, and infralimbic cortices of the mPFC regulated the valence of saccharin solution consumption from the rewarding property, the aversive property induced by morphine's conditioning, and the neutral states via saccharin extinction processes after morphine's conditioning. Methods: All rats received virus infection, buried optical fiber, optical stimulation, water deprivation, and saccharin solution consumption phases. In Experiment 1, rats were given ChR2 virus infection into the cingulate cortex (Cg1), prelimbic cortex (PrL), and infralimbic cortex (IL) to influence the rewarding saccharin solution consumption under photostimulation. In Experiment 2, rats were given ChR2 or EYFP virus infection into the Cg1, PrL, and IL to alter the saccharin solution consumption in the morphine-induced aversively conditioned taste aversion (CTA) and the saccharin solution consumption in the neutral state following the extinction process under photostimulation. Later, the immunohistochemical staining with c-Fos protein was performed for the Cg1, IL, PrL, nucleus accumbens core, nucleus accumbens shell, central amygdala, basolateral amygdala, ventral tegmental area, and dentate gyrus. Results: The results showed that optogenetic PrL stimulation decreased the rewarding valence of saccharin solution consumption and increased the morphine-induced, aversive valence of saccharin solution consumption. PrL stimulation decreased the neutral valence of saccharin solution consumption via the extinction process. Cg1 optogenetic stimulation increased the rewarding valence of saccharin solution consumption and the aversive valence of saccharin solution consumption induced by morphine in conditioning. Optogenetic IL stimulation increased the aversive valence of saccharin solution consumption induced by morphine via conditioning. Conclusion: Altogether, optogenetic stimulation in the subareas of the mPFC modulated the reward, aversion, and neutral valences of the stimulus and altered neuronal activity in the mPFC, amygdala, nucleus accumbens, and hippocampus. Notably, the change of valence was temporary alternation during light-on related to the light-off periods. However, the findings may provide insights in the development of novel treatments for addictive symptoms.

Neuronal activity of the medial prefrontal cortex, nucleus accumbens, and basolateral amygdala in conditioned taste aversion and conditioned place preference induced by different doses of morphine administrations in rats.

To re-examine the paradoxical effect hypothesis of abused drugs, the present study concerned whether different doses of morphine disparately affect neuronal activity and associations among the subareas of the medial prefrontal cortex (mPFC: cingulate cortex 1-Cg1, prelimbic cortex-PrL, infralimbic cortex-IL), the subregions of the nucleus accumbens (NAc; both core and shell), and the basolateral amygdala (BLA) following conditioned taste aversion (CTA) and conditioned place preference (CPP). All rats were given a 0.1% saccharin solution for 15-min, and they were intraperitoneally injected with saline or 20, 30, or 40 mg/kg morphine to form the aversive CTA learning. Later, half of the rats were tested for CPP (including the CTA and then CPP tests) for 30-min. Finally, the immunohistochemical staining with c-Fos was conducted after the behavioral test. After the CTA test, c-Fos (%) in the Cg1 and PrL (but not the IL) was more in 20-40 mg/kg of the morphine groups; c-Fos (%) in the NAc core, NAc shell, and BLA was more in the 30-40 mg/kg morphine group. After the CPP test, the Cg1, PrL, IL, and BLA showed more c-Fos (%) in 20 mg/kg morphine; the NAc core showed fewer in c-Fos (%) in the 30-40 mg/kg morphine groups. The mPFC subregions (e.g., Cg1, PrL, and IL), NAc subareas (e.g., NAc core and NAc shell), and BLA were involved in the different doses of morphine injections. The correlation analysis showed that a positive correlation was observed between PrL and IL with NAc core with low doses of morphine and with NAc shell with increasing doses of morphine after the CTA test. After the CPP, an association between PrL and NAc core and NAc shell at low doses and between IL and BLA and NAc shell with increasing doses of morphine. Therefore, different neural substrates and the neural connectivity are observed following different doses of morphine and after the CTA and CPP tests. The present data extend the paradoxical effect hypothesis of abused drugs.

The Medial Prefrontal Cortex, Nucleus Accumbens, Basolateral Amygdala, and Hippocampus Regulate the Amelioration of Environmental Enrichment and Cue in Fear Behavior in the Animal Model of PTSD.

A growing body of evidence showed that environmental enrichment (EE) ameliorated footshock-induced fear behavior of posttraumatic stress disorder (PTSD). However, no research comprehensively tested the effect of EE, cue, and the combination of EE and cue in footshock-induced fear behavior of PTSD symptoms. The present study addressed this issue and examined whether the medial prefrontal cortex (mPFC, including the cingulate cortex 1 (Cg1), prelimbic cortex (PrL), and infralimbic cortex (IL)), the nucleus accumbens (NAc), the basolateral amygdala (BLA), and the hippocampus (e.g., CA1, CA3, and dentate gyrus (DG)) regulated the amelioration of the EE, cue, or the combination of EE and cue. The results showed that EE or cue could reduce fear behavior. The combination of EE and cue revealed a stronger decrease in fear behavior. The cue stimulus may play an occasion setting or a conditioned stimulus to modulate the reduction in fear behavior induced by footshock. Regarding the reduction of the EE in fear behavior, the Cg1 and IL of the mPFC and the NAc upregulated the c-Fos expression; however, the BLA downregulated the c-Fos expression. The mPFC (i.e., the Cg1, PrL, and IL) and the hippocampus (i.e., the CA1, CA3, and DG) downregulated the c-Fos expression in the suppression of the cue in fear behavior. The interaction of EE and cue in reduction of fear behavior occurred in the Cg1 and NAc for the c-Fos expression. The data of c-Fos mRNA were similar to the findings of the c-Fos protein expression. These findings related to the EE and cue modulations in fear behavior may develop a novel nonpharmacological treatment in PTSD.

Involvement of the ventral tegmental area but not periaqueductal gray matter in the paradoxical rewarding and aversive effects of morphine.

The paradoxical effects of reward and aversion with abused drugs may interact to produce drug addiction, which is the so-called paradoxical effect hypothesis of abused drugs. However, there is no research examining how the ventral tegmental area (VTA) or periaqueductal gray matter (PAG) regulates morphine's paradoxical effect of reward and aversion. The present study addresses this issue, utilizing a high concentration of N-methyl-D-aspartic acid (NMDA) via injections to destroy the VTA or the PAG. Moreover, the study employed the new "pre- and postassociation" experimental paradigm (2010) to test whether the simultaneous rewarding and aversive effects of morphine can be affected by an NMDA lesion in the VTA or the PAG. The results indicated that the NMDA lesion of the VTA simultaneously reduced morphine-induced conditioned suppression of saccharin solution intake in conditioned taste aversion (CTA) and morphine-induced spent time in the preference compartment in conditioned place preference (CPP), whereas the PAG lesion did not change either measure. Thus, the VTA, but not the PAG, appears to contribute to the paradoxical effect reward in CPP and aversion in CTA induced by morphine. The VTA's involvement in morphine-induced CTA aversion and CPP reward supports the paradoxical effect hypothesis of abused drugs. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

BDNF Protein and BDNF mRNA Expression of the Medial Prefrontal Cortex, Amygdala, and Hippocampus during Situational Reminder in the PTSD Animal Model.

Whether BDNF protein and BDNF mRNA expression of the medial prefrontal cortex (mPFC; cingulated cortex area 1 (Cg1), prelimbic cortex (PrL), and infralimbic cortex (IL)), amygdala, and hippocampus (CA1, CA2, CA3, and dentate gyrus (DG)) was involved in fear of posttraumatic stress disorder (PTSD) during the situational reminder of traumatic memory remains uncertain. Footshock rats experienced an inescapable footshock (3 mA, 10 s), and later we have measured fear behavior for 2 min in the footshock environment on the situational reminder phase. In the final retrieval of situational reminder, BDNF protein and mRNA levels were measured. The results showed that higher BDNF expression occurred in the Cg1, PrL, and amygdala. Lower BDNF expression occurred in the IL, CA1, CA2, CA3, and DG. BDNF mRNA levels were higher in the mPFC and amygdala but lower in the hippocampus. The neural connection analysis showed that BDNF protein and BDNF mRNA exhibited weak connections among the mPFC, amygdala, and hippocampus during situational reminders. The present data did not support the previous viewpoint in neuroimaging research that the mPFC and hippocampus revealed hypoactivity and the amygdala exhibited hyperactivity for PTSD symptoms. These findings should be discussed with the previous evidence and provide clinical implications for PTSD.

Basolateral Amygdala but Not Medial Prefrontal Cortex Contributes to Chronic Fluoxetine Treatments for PTSD Symptoms in Mice.

Do chronic fluoxetine treatments reduced footshock-induced posttraumatic stress disorder (PTSD) symptoms, including fear and comorbid depression, in the situational reminder phase? Moreover, are the subareas of the medial prefrontal cortex (mPFC), including the cingulate cortex 1 (Cg1), prelimbic cortex (PrL), infralimbic cortex (IL), and basolateral amygdala (BLA), involved in the fluoxetine amelioration of PTSD symptoms? These two crucial issues were addressed in the present study. All mice were injected with chronic fluoxetine or normal saline treatments for the adaptation (14 days), footshock fear conditioning (1 day), and situational reminder (3 days) phases. After adaptation, the mice were subjected to footshock (2 mA, 10 seconds) or nonfootshock and stayed 2 min in a footshock box for 2 min for fear conditioning. Later, they were placed in the footshock box for 2 min in the situational reminder phase. In the final session of the situational reminder phase, a forced swimming test (FST) and immunohistochemical staining were conducted. The results indicated that footshock induced fear and comorbid depression. Meanwhile, chronic fluoxetine treatments reduced fear and depression behaviors. The Cg1, PrL, IL, and BLA were seemingly to increase c-Fos expression after footshock-induced PTSD symptoms in the situational reminder phase. The fluoxetine treatments reduced only the BLA's c-Fos expression. The findings suggest that BLA contributes to the fluoxetine amelioration of PTSD symptoms; however, the mPFC, including the Cg1, PrL, and IL, did not mediate PTSD symptoms' amelioration stemming from fluoxetine. The present data might help us to further understand the neural mechanism of fluoxetine treatments in PTSD symptoms.

Interactions between prelimbic cortex and basolateral amygdala contribute to morphine-induced conditioned taste aversion in conditioning and extinction.

The consequences of exciting or destroying the prelimbic cortex (PrL) or the basolateral amygdala (BLA) remain unclear, including the effects on morphine-induced conditioned taste aversion (CTA) in the conditioning and extinction phases, plasma corticosterone (CORT) levels, and c-Fos/p-ERK expressions in the subareas of the medial prefrontal cortex (i.e., PrL, infralimbic cortex [IL], cingulate cortex 1 [Cg1]), basolateral amygdala (BLA), central amygdala (CeA), hippocampus (i.e., CA1, CA2, CA3, and dentate gyrus [DG]), nucleus accumbens (NAc), lateral hypothalamus (LH), and piriform cortex (PC). During conditioning, excitation of the PrL glutamate neurons via NMDA injections disrupted morphine-induced CTA and decreased plasma CORT levels; moreover, c-Fos and p-ERK expression was hyperactive in the PrL and IL but hypoactive in the Cg1 and BLA. In conditioning, excitation of the BLA glutamate neurons via NMDA injections facilitated morphine-induced CTA and increased plasma CORT levels. The expression of c-Fos and p-ERK was hypoactive in the PrL and IL but hyperactive in the BLA. During extinction, lesion of the PrL glutamate neurons via NMDA injections impaired morphine-induced CTA extinction and enhanced plasma CORT levels. The expression of c-Fos and p-ERK was hypoactive in the PrL and IL but hyperactive in the BLA. In extinction, excitation of the PrL glutamatergic neurons via NMDA injections facilitated morphine-induced CTA extinction and did not affect plasma CORT levels; moreover, the expression of c-Fos and p-ERK was hypoactive in the Cg1, PrL, and IL but hyperactive in the BLA. Altogether, the interaction between the PrL and BLA plays a balancing role in morphine-induced CTA conditioning and extinction. During conditioning, the activity of the PrL correlated negatively with plasma CORT secretions, whereas the activity of the BLA correlated positively with the plasma CORT levels. During extinction, the activity of the PrL correlated negatively with plasma CORT secretions; however, the activity of the BLA may be negatively associated with the plasma CORT levels. The data presented here provide some implications for morphine addiction and dependence.

Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR.

Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (blaCTX-M-1 and blaCTX-M-2 families) and CREs (blaOXA-48 and blaKPC), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.

An ultrasensitive test for profiling circulating tumor DNA using integrated comprehensive droplet digital detection.

Current cancer detection systems lack the required sensitivity to reliably detect minimal residual disease (MRD) and recurrence at the earliest stages when treatment would be most effective. To address this issue, we present a novel liquid biopsy approach that utilizes an integrated comprehensive droplet digital detection (IC3D) digital PCR system which combines microfluidic droplet partitioning, fluorescent multiplex PCR chemistry, and our rapid 3D, large-volume droplet counting technology. The IC3D ddPCR assay can detect cancer-specific, ultra-rare genomic targets due to large sample input and high degree of partitioning. We first demonstrate our droplet digital PCR assay can robustly detect common cancer mutants including KRAS G12D spiked in wild-type genomic background or isolated from patient samples with 100% specificity. We then demonstrate that the IC3D ddPCR system can detect oncogenic KRAS G12D mutant alleles against a background of wild-type genomes at a sensitivity of 0.00125-0.005% with a false positive rate of 0% which is 50 to 1000× more sensitive than existing commercial liquid biopsy ddPCR and qPCR platforms, respectively. In addition, our technology can uniquely enable detection of circulating tumor cells using their genetic markers without a pre-enrichment step, and analysis of total tumor DNA isolated from blood samples, which will increase clinical sensitivity and specificity, and minimize inter-assay variability. Therefore, our technology holds the potential to provide clinicians with a powerful decision-making tool to monitor and treat MRD with unprecedented sensitivity for earlier stage intervention.

Distinct, genome-wide, gene-specific selectivity patterns of four glucocorticoid receptor coregulators.

Glucocorticoids are a class of steroid hormones that bind to and activate the glucocorticoid receptor (GR), which then positively or negatively regulates transcription of many genes that govern multiple important physiological pathways such as inflammation and metabolism of glucose, fat and bone. The remodeling of chromatin and regulated assembly or disassembly of active transcription complexes by GR and other DNA-binding transcription factors is mediated and modulated by several hundred transcriptional coregulator proteins. Previous studies focusing on single coregulators demonstrated that each coregulator is required for regulation of only a subset of all the genes regulated by a steroid hormone. We hypothesized that the gene-specific patterns of coregulators may correspond to specific physiological pathways such that different coregulators modulate the pathway-specificity of hormone action, thereby providing a mechanism for fine tuning of the hormone response. We tested this by direct comparison of multiple coregulators, using siRNA to deplete the products of four steroid hormone receptor coregulator genes (CCAR1, CCAR2, CALCOCO1 and ZNF282). Global analysis of glucocorticoid-regulated gene expression after siRNA mediated depletion of coregulators confirmed that each coregulator acted in a selective and gene-specific manner and demonstrated both positive and negative effects on glucocorticoid-regulated expression of different genes. We identified several classes of hormone-regulated genes based on the effects of coregulator depletion. Each coregulator supported hormonal regulation of some genes and opposed hormonal regulation of other genes (coregulator-modulated genes), blocked hormonal regulation of a second class of genes (coregulator-blocked genes), and had no effect on hormonal regulation of a third gene class (coregulator-independent genes). In spite of previously demonstrated physical and functional interactions among these four coregulators, the majority of the several hundred modulated and blocked genes for each of the four coregulators tested were unique to that coregulator. Finally, pathway analysis on coregulator-modulated genes supported the hypothesis that individual coregulators may regulate only a subset of the many physiological pathways controlled by glucocorticoids. We conclude that gene-specific actions of coregulators correspond to specific physiological pathways, suggesting that coregulators provide a potential mechanism for physiological fine tuning in vivo and may thus represent attractive targets for therapeutic intervention.

Coregulator cell cycle and apoptosis regulator 1 (CCAR1) positively regulates adipocyte differentiation through the glucocorticoid signaling pathway.

Glucocorticoids contribute to adipocyte differentiation by cooperating with transcription factors, such as CCAAT/enhancer-binding protein ß (C/EBPß), to stimulate transcription of the gene encoding peroxisome proliferator-activated receptor (PPARγ), a master regulator of adipogenesis. However, the mechanism of PPARγ gene regulation by glucocorticoids, the glucocorticoid receptor (GR), and its coregulators is poorly understood. Here we show that two GR binding regions (GBRs) in the mouse PPARγ gene were responsive to glucocorticoid, and treatment of 3T3-L1 preadipocytes with glucocorticoid alone induced GR occupancy and chromatin remodeling at PPARγ GBRs, which also contain binding sites for C/EBP and PPARγ proteins. GR recruited cell cycle and apoptosis regulator 1 (CCAR1), a transcription coregulator, to the PPARγ gene GBRs. Notably, CCAR1 was required for GR occupancy and chromatin remodeling at one of the PPARγ gene GBRs. Moreover, depletion of CCAR1 markedly suppressed differentiation of mouse mesenchymal stem cells and 3T3-L1 preadipocytes to mature adipocytes and decreased induction of PPARγ, C/EBPα, and C/EBPδ. Although CCAR1 was required for stimulation of several GR-regulated adipogenic genes in 3T3-L1 preadipocytes by glucocorticoid, it was not required for GR-activated transcription of certain anti-inflammatory genes in human A549 lung epithelial cells. Overall, our results highlighted the novel and specific roles of GR and CCAR1 in adipogenesis.

Recruitment of coregulator G9a by Runx2 for selective enhancement or suppression of transcription.

Runx2, best known for its role in regulating osteoblast-specific gene expression, also plays an increasingly recognized role in prostate and breast cancer metastasis. Using the C4-2B/Rx2(dox) prostate cancer cell line that conditionally expressed Runx2 in response to doxycycline treatment, we identified and characterized G9a, a histone methyltransferase, as a novel regulator for Runx2 activity. G9a function was locus-dependent. Whereas depletion of G9a reduced expression of many Runx2 target genes, including MMP9, CSF2, SDF1, and CST7, expression of others, such as MMP13 and PIP, was enhanced. Physical association between G9a and Runx2 was indicated by co-immunoprecipitation, GST-pulldown, immunofluorescence, and fluorescence recovery after photobleaching (FRAP) assays. Since G9a makes repressive histone methylation marks and is primarily known as a corepressor, we further investigated the mechanism by which G9a functioned as a positive regulator for Runx2 target genes. Transient reporter assays indicated that the histone methyltransferase activity of G9a was not required for transcriptional activation by Runx2. Chromatin immunoprecipitation assays for Runx2 and G9a showed that G9a was recruited to endogenous Runx2 binding sites. We conclude that a subset of cancer-related Runx2 target genes require recruitment of G9a for their expression, but do not depend on its histone methyltransferase activity.

Reciprocal roles of DBC1 and SIRT1 in regulating estrogen receptor α activity and co-activator synergy.

Estrogen receptor α (ERα) plays critical roles in development and progression of breast cancer. Because ERα activity is strictly dependent upon the interaction with coregulators, coregulators are also believed to contribute to breast tumorigenesis. Cell Cycle and Apoptosis Regulator 1 (CCAR1) is an important co-activator for estrogen-induced gene expression and estrogen-dependent growth of breast cancer cells. Here, we identified Deleted in Breast Cancer 1 (DBC1) as a CCAR1 binding protein. DBC1 was recently shown to function as a negative regulator of the NAD-dependent protein deacetylase SIRT1. DBC1 associates directly with ERα and cooperates synergistically with CCAR1 to enhance ERα function. DBC1 is required for estrogen-induced expression of a subset of ERα target genes as well as breast cancer cell proliferation and for estrogen-induced recruitment of ERα to the target promoters in a gene-specific manner. The mechanism of DBC1 action involves inhibition of SIRT1 interaction with ERα and of SIRT1-mediated deacetylation of ERα. SIRT1 also represses the co-activator synergy between DBC1 and CCAR1 by binding to DBC1 and disrupting its interaction with CCAR1. Our results indicate that DBC1 and SIRT1 play reciprocal roles as major regulators of ERα activity, by regulating DNA binding by ERα and by regulating co-activator synergy.

A coactivator role of CARM1 in the dysregulation of ß-catenin activity in colorectal cancer cell growth and gene expression.

Aberrant activation of Wnt/ß-catenin signaling, resulting in the expression of Wnt-regulated oncogenes, is recognized as a critical factor in the etiology of colorectal cancer. Occupancy of ß-catenin at promoters of Wnt target genes drives transcription, but the mechanism of ß-catenin action remains poorly understood. Here, we show that CARM1 (coactivator-associated arginine methyltransferase 1) interacts with ß-catenin and positively modulates ß-catenin-mediated gene expression. In colorectal cancer cells with constitutively high Wnt/ß-catenin activity, depletion of CARM1 inhibits expression of endogenous Wnt/ß-catenin target genes and suppresses clonal survival and anchorage-independent growth. We also identified a colorectal cancer cell line (RKO) with a low basal level of ß-catenin, which is dramatically elevated by treatment with Wnt3a. Wnt3a also increased the expression of a subset of endogenous Wnt target genes, and CARM1 was required for the Wnt-induced expression of these target genes and the accompanying dimethylation of arginine 17 of histone H3. Depletion of ß-catenin from RKO cells diminished the Wnt-induced occupancy of CARM1 on a Wnt target gene, indicating that CARM1 is recruited to Wnt target genes through its interaction with ß-catenin and contributes to transcriptional activation by mediating events (including histone H3 methylation) that are downstream from the actions of ß-catenin. Therefore, CARM1 is an important positive modulator of Wnt/ß-catenin transcription and neoplastic transformation, and may thereby represent a novel target for therapeutic intervention in cancers involving aberrantly activated Wnt/ß-catenin signaling.

Requirement of cell cycle and apoptosis regulator 1 for target gene activation by Wnt and beta-catenin and for anchorage-independent growth of human colon carcinoma cells.

Aberrant Wnt signaling promotes oncogenesis by increasing cellular levels of beta-catenin, which associates with DNA-bound transcription factors and activates Wnt target genes. However, the molecular mechanism by which beta-catenin mediates gene expression is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which was recently shown to function as a transcriptional coactivator for nuclear receptors, also interacts with beta-catenin and enhances the ability of beta-catenin to activate expression of transiently transfected reporter genes. Furthermore, association of CCAR1 with the promoter of an endogenous Wnt/beta-catenin target gene in a colon cancer cell line depends on the presence of beta-catenin. Depletion of CCAR1 inhibits expression of several Wnt/beta-catenin target genes and suppresses anchorage-independent growth of the colon cancer cell line. Thus, CCAR1 is a novel component of Wnt/beta-catenin signaling that plays an important role in transcriptional regulation by beta-catenin and that, therefore, may represent a novel target for therapeutic intervention in cancers involving aberrantly activated Wnt/beta-catenin signaling.

Role of GAC63 in transcriptional activation mediated by beta-catenin.

Beta-catenin is a key mediator in the canonical Wnt signaling pathway, which plays important roles in multiple developmental processes. Inappropriate activation of this pathway leads to developmental defects and development of certain cancers. Upon Wnt signaling, beta-catenin binds TCF/LEF transcription factors. The TCF/LEF-beta-catenin complex then recruits a variety of transcriptional coactivators to the promoter/enhancer region of Wnt-responsive genes and activates target gene transcription. In this article, we demonstrate that GRIP1-associated coactivator 63 (GAC63), a recently identified nuclear receptor (NR) coactivator, interacts with beta-catenin. The N-terminus of GAC63 is the binding site for beta-catenin, whereas a C-terminal fragment of beta-catenin including armadillo repeats 10-12 binds to GAC63. Over-expression of GAC63 enhanced the transcriptional activity of beta-catenin, and also greatly enhanced TCF/LEF-regulated reporter gene activity in a beta-catenin-dependent manner. Endogenous GAC63 was recruited to TCF/LEF-responsive enhancer elements when beta-catenin levels were induced by LiCl. In addition, reduction of endogenous GAC63 level by small interfering RNA (siRNA) inhibited TCF/LEF-mediated gene transcription. Our findings reveal a new function of GAC63 in transcriptional activation of Wnt-responsive genes.

Transcriptional intermediary factor 1alpha mediates physical interaction and functional synergy between the coactivator-associated arginine methyltransferase 1 and glucocorticoid receptor-interacting protein 1 nuclear receptor coactivators.

In previous studies transcriptional intermediary factor 1alpha (TIF1alpha) was identified as a direct binding partner and potential transcriptional coactivator for nuclear receptors (NRs) but its overexpression inhibited, rather than enhanced, transcriptional activation by NRs. Here we show that TIF1alpha bound to and enhanced the function of the C-terminal activation domain (AD) of coactivator associated arginine methyltransferase 1 (CARM1) and the N-terminal AD of glucocorticoid receptor-interacting protein 1 (GRIP1). Furthermore, although TIF1alpha had little or no NR coactivator activity by itself, it cooperated synergistically with GRIP1 and CARM1 to enhance NR-mediated transcription. Inhibition of endogenous TIF1alpha expression reduced transcriptional activation by the GRIP1 N-terminal domain but not by the CARM1 C-terminal domain, suggesting that TIF1alpha may be more important for mediating the activity of the former than the latter. Reduction of endogenous TIF1alpha levels also compromised the androgen-dependent induction of an endogenous target gene of the androgen receptor. Finally, TIF1alpha formed a ternary complex with the GRIP1 N-terminal and CARM1 C-terminal domains. Thus, we conclude that TIF1alpha cooperates with NR coactivators GRIP1 and CARM1 by forming a stable ternary complex with them and enhancing the AD function of one or both of them.